The present invention is in the fields of molecular biology, protein chemistry and protein purification. Specifically, the invention provides compositions comprising reverse transcriptases (RTs) and methods for the production of such reverse transcriptase enzymes. Such methods provide for reverse transcriptases that are substantially free from contamination by nucleic acids and other unwanted materials or proteins. Compositions comprising the reverse transcriptase enzymes of the present invention may be used in a variety of applications, including synthesis, amplification and sequencing of nucleic acids.
A variety of techniques may be employed to facilitate the preparation of intracellular proteins from microorganisms. Typically, the initial steps in these techniques involve lysis or rupture of the bacterial cells, to disrupt the bacterial cell wall and allow release of the intracellular proteins into the extracellular milieu. Following this release, the desired proteins are purified from the extracts, typically by a series of chromatographic steps.
Several approaches have proven useful in accomplishing the release of intracellular proteins from bacterial cells. Included among these are the use of chemical lysis, physical methods of disruption, or a combination of chemical and physical approaches (reviewed in Felix, H., Anal. Biochem. 120:211-234 (1982)).
Chemical methods of disruption of the bacterial cell wall that have proven useful include treatment of cells with organic solvents such as toluene (Putnam, S. L., and Koch, A. L., Anal. Biochem. 63:350-360 (1975); Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982)), with chaeotropes such as guanidine salts (Hettwer, D., and Wang, H., Biotechnol. Bioeng. 33:886-895 (1989)), with antibiotics such as polymyxin B (Schupp, J. M., et al., BioTechniques 19:18-20 (1995); Felix, H., Anal. Biochem. 120:211-234 (1982)), or with enzymes such as lysozyme or lysostaphin (McHenty, C. S., and Kornberg, A., J. Biol. Chem. 252(18):6478-6484 (1977); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990); Hughes, A. J., Jr., et al., J. Cell Biochem. Suppl. 016 (Part B):84 (1992); Sambrook, J., et al., in Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.; Cold Spring Harbor Laboratory Press, p. 17-38 (1989); Ausubel, F. M., et al.; in Current Protocols in Molecular Biology, New York: John Wiley and Sons, p. 4.4.1-4.47 (1993)). The effects of these various chemical agents may be enhanced by concurrently treating the bacterial cells with detergents such as Triton X-100(copyright), sodium dodecylsulfate (SDS) or Brij 35 (Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982); Hettwer, D., and Wang, H., Biotechnol. Bioeng, 33:886-895 (1989); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990); Schupp, J. M., et al., BioTechniques 19:18-20 (1995)), or with proteins or protamines such as bovine serum albumin or spermidine (McHenry, C. H. and Kornberg, A., J. Biol. Chem. 252(18): 6478-6484(1977); Felix, H., Anal. Biochem. 120:211-234 (1982); Hughes, A. J., Jr., et al., J. Cell Biochem. Suppl. 0 16 (Part B):84 (1992)).
In addition to these various chemical treatments a number of physical methods of disruption have been used. These physical methods include osmotic shock, e.g., suspension of the cells in a hypotonic solution in the presence or absence of emulsifiers (Roberts, J. D., and Lieberman, M. W., Biochemistry 18:4499-4505 (1979); Felix, H., Anal. Biochem. 120:211-234 (1982)), drying (Mowshowitz, D. B., Anal. Biochem. 70:94-99 (1976)), bead agitation such as ball milling (Felix, H., Anal. Biochem. 120:211-234 (1982); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:182:147-153 (1990)), temperature shock, e.g., freeze-thaw cycling (Lazzarini, R. A., and Johnson L. D., Nature New Biol. 243:17-20 (1975); Felix, H., Anal, Biochem. 120:211234 (1982)), sonication (Amos, H., et al., J. Bacteriol. 94:232-240 (1967); Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York:John Wiley and Sons, pp. 4.4.1-4.47 (1993)) and pressure disruption, e.g., use of a french pressure cell (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York:John Wiley and Sons, pp. 16.8.6-16.8.8 (1993)). Other approaches combine these chemical and physical methods of disruption, such as lysozyme treatment followed by sonication or pressure treatment, to maximize cell disruption and protein release (Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York:John Wiley and Sons, pp. 4.4.1-4.47 (1993)).
These disruption approaches have several advantages, including their ability to rapidly and completely (in the case of physical methods) disrupt the bacterial cell such that the release of intracellular proteins is maximized. In fact, these approaches have been used in the initial steps of processes for the purification of a Variety of bacterial cytosolic enzymes, including natural and recombinant proteins from mesophilic organisms such as Escherichia coli, Bacillus subtilis and Staphylococcus aureus (Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977); Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990); Hughes, A. J., Jr., et al., J. Cell Biochem. Suppl. 0 16 (Part B): 84 (1992); Ausubel, F. M., et al., in Current Protocols in Molecular Biology, New York: John Wiley and Sons, pp. 4.4.1-4.47 (1993)), as well as phosphatases, restriction enzymes, DNA or RNA polymerases and other proteins from thermophilic bacteria and archaea.
However, these methods possess distinct disadvantages as well. For example, the physical methods by definition involve shearing and fracturing of the bacterial cell walls and plasma membranes. These processes thus result in extracts containing large amounts of particulate matter, such as membrane or cell wall fragments, which must be removed from the extracts, typically by centrifugation, prior to purification of the enzymes. This need for centrifugation limits the batch size capable of being processed in a single preparation to that of available centrifuge space; thus, large production-scale preparations are impracticable if not impossible. Furthermore, physical methods, and may chemical techniques, typically result in the release from the cells not only of the desired intracellular proteins, but also of undesired nucleic acids and membrane lipids (the latter particularly resulting when organic solvents are used). These undesirable cellular components also complicate the subsequent processes for purification of the desired proteins, as they increase the viscosity of the extracts (Sambrook, J., et al., in: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p. 17-38 (1989); Cull, M., and McHenry, C. S., Meth. Enzynol. 182:147-153 (1990)), and bind with high avidity and affinity to nucleic acid-binding proteins such as DNA polymerases, RNA polymerases and restriction enzymes.
One problem associated with these approaches is that the enzyme preparations are typically contaminated with nucleic acids (e.g., RNA and DNA). This contaminating nucleic acid may come not only from the organisms which are the source of the enzyme, but also from unknown organisms present in the reagents and materials used to purify the enzyme after its release from the cells. Since reverse transcriptase enzymes are routinely used in techniques of amplification and synthesis of nucleic acid molecules (e.g., the Polymerase Chain Reaction (PCR), particularly RT-PCR) the presence of contaminating DNA or RNA in the enzyme preparations is a significant problem since it can give rise to spurious amplification or synthesis results. Thus, a need exists for preparation of reverse transcriptase enzymes that are substantially free of contamination by nucleic acids.
Instead of attempting to remove nucleic acids from preparations of reverse transcriptase enzymes, a more reasonable and successful approach would be to prevent contamination of the enzymes by nucleic acids from the outset in the purification process. Such an approach would be two-pronged: 1) preventing release of nucleic acids from the bacterial cells during permeabilization of the cells to release the enzymes; and 2) preventing contamination of the enzymes during the purification process itself. Furthermore, an optimal method would obviate the need for centrifugation in the process, thus allowing large-scale, and even continuous, production of nucleic acid-free reverse transcriptase enzymes. The present invention provides such methods, and reverse transcriptase enzymes produced by these methods.
The present invention generally provides methods of making a reverse transcriptase enzyme comprising permeabilizing a cellular source of reverse trans criptase (e.g., bacterial cells) to form spheroplasts or protoplasts and isolating the reverse transcriptase enzyme. Preferably, the methods are conducted under conditions favoring the partitioning of nucleic acids from the reverse transcriptase enzyme. In particular, the invention relates to a method for isolation or purification of reverse transcriptases comprising cell permeabilization, filtration and isolation.
The invention is particularly directed to methods wherein the permeabilization of the cells is accomplished by contacting the cells with an aqueous solution comprising at least one of: a chaeotropic agent, preferably a guanidine salt and most preferably guanidine hydrochloride; and/or a nonionic detergent, preferably Triton X-100 and/or sodium deoxycholic acid. The invention is further directed to such methods wherein the conditions favoring the partitioning of nucleic acids from the reverse transcriptase enzyme comprise formation of an filtrate (e.g., ultrafiltrate) by filtration (e.g., microfiltration) of the cellular source subjected to permeabilization (particularly of the spheroplasts or protoplasts) through a semi-permeable membrane, which is preferably a hydrophilic dialysis membrane, preferably in the presence of a salt, preferably ammonium sulfate, and purification or isolation of the reverse transcriptase enzyme from the filtrate, preferably by chromatography using sterile materials. The invention is particularly directed to such methods wherein bacterial cells providing the reverse transcriptase enzyme are used, preferably prokaryotic cells such as those of species of the genera Escherichia (preferably E. coli), Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Klebsiella, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, Xanthomonas and Streptomyces. In another aspect, the cellular source of reverse transcription is a recombinant cellular source.
The invention also provides the reverse transcriptase enzymes, or mutants, derivatives or fragments thereof, that are made according to the methods provided. The invention is also directed to methods for amplifying or synthesizing a nucleic acid molecule comprising contacting a nucleic acid molecule (e.g., template) with an reverse transcriptase made according to the methods of the present invention under conditions to make a first nucleic acid molecule complementary to all or a portion of the template. Such synthesis or amplification may further comprise incubating the reaction with one or more polymerases (DNA polymerases, preferably thermostable DNA polymerases such as Tne, Tma, Taq etc. or mutants, derivatives or fragments thereof) under conditions sufficient to make a second nucleic acid molecule complementary to all or a portion of the first nucleic acid molecule.
The invention also provides kits for amplifying or synthesizing nucleic acid molecules comprising a carrier means having in close confinement therein one or more container means, wherein said kit may comprise at least one component selected from one or more reverse transcriptases produced according to the invention, one or more polymerases (e.g., DNA polymerases), one or more nucleotides or derivatives thereof, one or more primers, and one or more synthesis or amplification reaction buffers.
Other features and advantages of the present invention will be apparent to those skilled in the art from the following description of the preferred embodiments and from the claims.